Supplemental Content 1: Supplemental Materials and Methods
MATERIALS AND METHODS
Animals
All procedures and animal care issues in this study were approved by the institutional
Animal Care and Use Committee and followed DHHS guidelines. Female domestic pigs
weighing 22 to 33kg were procured and allowed to acclimate to housing conditions. Surgeries
were performed in a large animal surgery laboratory by the same experienced investigators and
animals were individually housed in large animal care facilities.
For all procedures, the animals were initially sedated with intramuscular Ketamine (18-22
mg/kg): Acepromazine (0.8-1.2 mg/kg): Xylazine [0.8-1.2 mg/kg]. Animals were intubated and
anesthesia maintained with 1.5 to 2.0% Isoflurane by inhalation during the preparation, impact
procedure, and MRI-CT scans. A ventilator (Drager, North America, Ave, Telford,
Pennsylvania, PA) was used to deliver inhalation anesthetic during creation of the CCI.
Heart/respiratory rates, blood pressure, blood oxygen, and the end tidal C02 concentration was
monitored continuously (SurgiVent, Duplin, OH).
Uninjured Brain Blood flow and Deformation Experiments
A craniectomy was performed with a manual trephine straddling the right coronal suture
at the junction of the superior temporal line and the orbital rim and enlarged to 4cm by 3cm. The
dura was removed to expose brain. Bipolar cautery was used as necessary to achieve hemostasis
of the dura.
Compound matrix consisting of a fine pore non-adherent synthetic sheet interface was
placed directly on the brain. As opposed to standard negative pressure wound therapy matrices,
this interface was chosen to prevent ingrowth and adherence of brain tissue into the matrix as
well as to prevent the stimulation of granulation tissue on the surface of the brain. Overlying this
was placed a second matrix which is a reticulated open cell polyurethane matrix with larger
interstices to facilitate the removal of fluid and minimize the risk of coagulation of proteinacious
material. This matrix was connected by a flexible silicone tube to a receptacle attached to a
computerized vacuum pump. The matrix was held in place and a controlled sealed wound
created by resuturing of the overlying skin. It is critical that the overlying skin be closed so that a
negative pressure can be maintained. Leakage from inability to obtain an air tight seal will result
in desiccation and loss of vacuum. If necessary, an adhesive polyethylene sheet can be used to
insure closure. All wounds in this experiment were closed primarily without the use of
reinforcing sheets. Animals could move freely in the cage with this device in place.
Traumatic Brain Injury (CCI) Experiments
The studies involving traumatic brain injury procedures were performed following
previous models. A midline incision was made over the skull, a 17-mm bur hole was made on
the right side of the skull, 7 mm anterior to the coronal suture and 3 mm lateral to the midline
over the frontal lobe to expose the dura over the frontal cortex for impact. The head of the
animal was placed in a modified large-animal stereotactic frame to prevent movement at the time
of impact. The traumatic brain injury (controlled cortical impact, CCI) was induced with a CCI
device (Model AMS 201, AmScien Instruments, Richmond, VA).
The 12 mm diameter
impactor tip with beveled edge was centered in the craniectomy site perpendicular to the exposed
surface of the brain. Gas pressure of 60 psi was used, giving an impact velocity of 2.7 m/s and
duration of 250 ms to produce a brain deformation of 12 mm during impact. Impact resulted in
injuries including dura laceration, CSF leaking, contusion of the underlying brain with
hemorrhage. A vacuum drainage dressing as previously described as then placed. All pigs were
randomized to their study groups after CCI. A maximum of 5-12cc of serosangunious fluid with
minimal blood content was removed by the device in all treated animals. No large volumes of
CSF or blood were noted. No brain tissue was found in the fluid removed. There was neither
disruption of cerebral vessels nor distortion of bridging veins.
Intracranial Pressure Monitoring and Telemetry Recording
In the CCI groups of animals, a 3.5 mm diameter burhole was made on the left side of the
skull, 4mm a lateral to midline and 4 mm posterior bregma, and intracranial pressures were
monitored by inserting PA-C40 pressure probe (DSI, Data Science International, St. Paul, MN).
The ICP data were collected every 30 min. No interference from animal activity was apparent.
A dual pressure capable D70-PCTP transmitter (DSI) was implanted in the left neck to
monitor physiologic parameters. One pressure catheter was inserted for ICP monitoring, another
catheter into left radial artery for continuous blood pressure measurement. Two biopotential
probes were connected to stainless screws across the craniotomy for EEG recording. The mean
ICP, blood pressure, temperature, heart rate, and animal activity were collected from recorded
data. EEG biopotential analysis was performed by Neuroscore (DSI). Seizures were defined as a
sudden onset of high amplitude (>2× background) activity with a duration greater than 10
seconds. Animals were also scored for periods of epileptiform activity that could not be strictly
defined as seizures. ICP and biopotential probes were removed to allow MRI on the third day
after surgery and not replaced.
CT perfusion
Blood flow was determined by CT perfusion studies. These studies, while not directly
necessary blood flows, are accepted as noninvasive reproducible measurements of relative blood
flow. Heart rate, respiration rate, oxygen saturation, and end tidal Isoflurane and C02
concentrations were monitored and maintained. End tidal carbon-dioxide was maintained
between 34-37mm Hg. CT perfusion was performed in GE Discovery PET/CT 64 Slice after
administration of a contrast bolus Omnipaque 350 (2ml/kg) with an injection rate of 4 mL/s
delivered via power-injector. Scan and contrast injection started at same time. Scan parameters
were used in Cine Scan: 40mm Detector coverage, 1sec rotation time, 5 mm slice thickness, 8
images per rotation, 80kV, 200mA, DFOV 25, Standard Reconstruction, total exposure time of
45 sec. 8 contiguous sections from surface of skull to the level of the basal ganglia were
imaged. The pericollasal artery was chosen as artery input for TeraRecon analysis and CBF,
CBV, and MTT data of 4 pigs were collected. The venous out flow was obtained from the
superior sagittal sinus.
Calculation of perfusion parameters and maps was performed with a commercial
perfusion package (Aquarius Intuition Viewer version4.4.7, TeraRecon, Foster City, CA).
MRI Procedures
MRI was performed 3 days post injury with a GE (Milwaukee, WI) Signa EchoSpeed
1.5-T scanner after ICP and biopotential probes were removed for Aim 1, determination of level
of applied negative pressure. MRI was performed 5 days post injury with a GE (Milwaukee, WI)
Signa EchoSpeed 1.5-T scanner after ICP and biopotential probes were removed for Aim 2,
determination of optimal length of time that MTR should be applied to injured brain. MRI was
performed 5 days post injury with a GE (Milwaukee, WI) Signa EchoSpeed 1.5-T scanner or a
3TSiemens SKYRA MRI (Malvern, PA) after ICP and biopotential probes were removed for
Aim 3, the effect of time-delay of treatment after the initial controlled cortical injury (CCI).
Animals were maintained under Isoflurane anesthesia and placed in an 8-channel HR Brain coil.
Localizer scans and anatomic images with the Signa EchoSpeed 1.5-T scanner were collected
using the routine head protocol for coronal T2 fast spin-echo (FSE-XL, TE 84.82 msec, TR 8000
msec, slice thickness/gap 2/0 mm, field of view [FOV] 13 cm, matrix 128 × 128, number of
excitations [NEX] 4, scan time was 6 minutes). T2*-weighted gradient-echo MR imaging was
used in the detection of intracerebral hemorrhage. Coronal MPGR (Multi-Planar Gradient Echo,
TR 616 msec, TE 11 msec, flip angle 15 degrees, thickness/gap 2 / 0 mm, FOV 13x13 cm,
matrix 128 x 128, NEX 2, scan time was 5 min 23 sec) was performed. Similar parameters were
used for the 3T Siemens MRI.
MRI was performed for normal, non-injured pigs with 3D BRAVO procedures (TR 12.04
msec, TE 5.02 msec, TI 600 msec, thickness 0.7 mm) and all MRI measurements were
performed on a TeraRecon workstation (Foster City, CA). Total contusion injured brain volumes
were measured in all coronal MR T2 weighted images as the sum of all injury areas in both
groups. The injured area was identified and traced as a hyperintense region ipsilateral to the
injured site. There was a large area of T2 hyperintensity (edema) sometimes associated with
hypointensity (hemorrhage) and herniation in T2-weighted images. Total hemorrhage area as
hypointensity was measured in gradient echo images.
Proton-magnetic resonance spectra were obtained post-injury from a 10 mm3 voxel using
point-resolved spectroscopy with the following parameters: TR 1500 ms, TE 35 ms, 90° angle
and 190 averages. All MR spectroscopy (MRS) data were processed and analyzed using a
Linear Combination (LC) model. While there are limitations on quantifying metabolites in neural
tissue, MRI spetra are accepted as reproducible, non-invasive relative quantity indicators.
Histology
All pigs were euthanized and immediately perfused with 4% paraformaldehyde through
the ascending aorta eight days after CCI. The brain was removed and postfixed in the same
fixative solution overnight at 4ºC, rinsed in PBS, then placed in 30% sucrose, then were snapfrozen in O.C.T and stored at -80ºC. Coronal sections of injured area were cut into 50 μm in
thickness using a cryostat, mounted, and kept frozen until use. Sections were collected every 1.6
mm through all injured area over 1.7 cm. Sections were examined after staining with
haematoxylin and eosin (H&E). The cerebral contused/damaged volumes were measured by
gross pathological examination in each coronal section. Each block containing the contusion
lesion was photographed and transferred to a personal computer for analysis. The identified
lesion boundaries in the gross specimens were defined as areas of necrosis and hemorrhage using
ImageJ (NIH) software.
Statistical Analysis
Statistical analysis was performed for several different groups such as non-treated and
treated post-CCI. For ICP, neuroimaging analysis data, comparisons of groups and time points
were made using two-way repeated measures ANOVA with Student-Newman-Keuls post-hoc
tests or student T-tests.